In some countries sampling of amniotic fluid is offered to pregnant women having an increased risk of bearing children with chromosome disorders. If the sample collected indicates that the child to be born is diseased an induced abortion is offered.
With the presently applied sampling technique 15-20 ml amniotic fluid is collected in the 16th week of pregnancy, and normally a further 3-4 weeks elapse before a result of the laboratory examination is available. Consequently, the pregnant woman in most cases will be in the 19th-20th week of pregnancy before an abortion can be induced. Of course this represents a heavy strain for the woman at this advanced stage of pregnancy and also from a medical point of view it is inappropriate.
With the present technique it is not possible to carry out the sampling of amniotic fluid earlier, since this involves a considerable risk that the collected sample fails to give the desired result due to a too low content of cells. When routine amniocentesis is carried out approximately 10% of the total volume of the amniotic fluid is collected. It has been shown that if more than 17 ml is collected in the 16th week of pregnancy, the risk of respiratory distress in the neonatals is increased (Jo-Anne K. Finegan, 1984). Thus, the motivation for collecting samples at this late stage of pregnancy has been to achieve a successful culturing of cells and partly to avoid the risk of spontaneous abortion which risk is known to be relatively low at this stage (A. Tabor, Thesis, 1988).
During recent years, chorionic villi biopsy has been developed in order to obtain early chromosomal examinations of the fetus. By this technique, it is normally possible to obtain a result in the late first trimester. However, this method of sampling involves the problem that the placenta tissue is not always chromosomally identical with the fetus which is a prerequisite for a correct diagnosis (Verjaal M. et al., 1987; Eiben B. et al., 1989). Consequently, it is necessary to carry out amniocentesis in a significant number of pregnant women in the 16th pregnancy week in order to determine whether the diagnosis is correct or not, since amniocentesis is still the most reliable method for chromosomal examinations of fetuses. Furthermore, chorionic villi biopsy is an expensive examination. It is estimated that a chorionic villi biopsy sample requires the double number of technician hours to be processed when compared to an amniotic fluid sample.
It is therefore of considerable relevance to improve the amniocentesis technique. Preliminary results from USA indicate that amniocentesis carried out at an early stage, even as early as the 9th week of pregnancy, implies a low risk of spontaneous abortion, and that the risk of complications at later stages of the pregnancy does not seem to be increased (Hanson F. W. et al., 1987; Elejalde B. R. and Elejalde M. M., 1988; Goodmilow L. et al., 1988).
With the presently used technique, only the cells in a sample of amniotic fluid are used for chromosomal fetus examination. After centrifugation of the sample, the supernatant is discarded, so that only the cells remain. Subsequently, these cells are cultivated for 2-3 weeks in order to obtain a sufficient number of dividing cells for the chromosomal examination. Only 2 ml of amniotic fluid is used for alpha-fetoprotein determination, and this part of the amniocentesis is independent of the concentration of cells. The cells in the amniotic fluid are considered to be waste products without any significance for the development of the fetus.
The present invention provides a method of the above type and the method according to the invention is characterized in separating outside the amniotic cavity amniotic fluid with a reduced content of cells from the extracted volume of amniotic fluid and recirculating at least part of the separated fluid to the amniotic cavity substantially immediately after the separation, whereby a sample with an increased cell concentration is retained.
Because the separated amniotic fluid with reduced content of cells is substantially immediately returned to the amniotic cavity, a sample containing the necessary amount of cells may be collected by permanently removing only a relatively small volume of amniotic fluid from the amniotic cavity. Because of the relatively small volume of amniotic fluid necessary the sample collection can be carried out at an earlier stage of pregnancy without an increase of the abortion risk and even with a reduced risk of abortion.
Since the cells in the amniotic fluid are considered to be waste products which may be removed from the amniotic cavity without any risk increase the method according to the invention renders it possible to collect a sample containing a relatively large amount of cell material. This means that the necessary time for carrying out the laboratory examination may be reduced, because the cell material may be cultivated in a time period which is shorter than hitherto.
It is primarily the relative amount of amniotic fluid being removed from the amniotic cavity which is decisive for how early in the pregnancy period a sample can be collected. In the method according to the invention the amniotic fluid removed from the amniotic cavity may be separated continuously and the separated amniotic fluid with its reduced cell content may then simultaneously and continuously be returned to the amniotic cavity through a return passage. This means that in principle a rather considerable part of the cell material present within the amniotic cavity may be removed therefrom while only a rather small amount of amniotic fluid is present outside the amniotic cavity at any time.
Alternatively the volume of amniotic fluid may be withdrawn from the amniotic cavity in two or more part volumes and after the withdrawal of each of these volumes the separation and the subsequent recirculation of amniotic fluid to the amniotic cavity may be take place before the next part volume is withdrawn or taken out. Consequently only one part volume is removed from the amniotic cavity at a time and the amount of fluid present in such part volume may then be adapted to the total amount of amniotic fluid present in the amniotic cavity at the time when the sample is to be taken out, whereby the risk may be reduced to an acceptable level.
The separation of amniotic fluid with a reduced content of cells may be performed in any suitable manner, for example by centrifuging. However, the separation is preferably performed by filtration by passing at least part of the volume of amniotic fluid withdrawn from the amniotic cavity through a cell filter which may be arranged at a suitable position within the flow passage of the amniotic fluid outside the amniotic cavity. Thus, part of the cells present in the amniotic fluid will be filtered from the fluid and be retained by the filter. The amniotic fluid having flown through the filter whereby its content of cells has been reduced may then continuously or intermittently be returned to the amniotic cavity. The cells which are filtered from the amniotic fluid and which are deposited on the filter may be removed together with the cell filter and subsequent cultivation of the cells may take place on the filter. Such a procedure involves a substantial saving of time and rationalization in cultivating the cell material.
The volume of amniotic fluid may be withdrawn from the amniotic cavity in any suitable manner. As an example, the amniotic fluid may be continuously withdrawn from the amniotic cavity and recirculated thereto through an external flow passage through which the fluid is circulated by means of a peristaltic pump or another kind of pump In the preferred embodiment, however, the volume of amniotic fluid is withdrawn from the amniotic cavity by means of a syringe, and amniotic fluid is then drawn or sucked into a chamber defined within the syringe and thereafter displaced from the syringe chamber and returned to the amniotic cavity, the amniotic fluid being passed through a cell filter when the fluid is drawn into or displaced from the syringe chamber. This means that the syringe should be adapted so as to cause the amniotic fluid to flow through the cell filter mainly in one direction.
The invention also relates to an apparatus or sampling device for use in carrying out the method as described above and comprising a hollow needle or cannula for penetrating the wall of the amniotic cavity, a sample receiving chamber communicating with the hollow needle or cannula, and means for drawing a volume of amniotic fluid into the sample receiving chamber through the hollow needle, and the apparatus according to the invention is characterized in that the apparatus further comprises separating means for separating amniotic fluid with a reduced content of-cells from said volume of amniotic fluid drawn into the sample receiving chamber, and fluid returning means for returning at least part of the separated amniotic fluid to the amniotic cavity.
The apparatus or sampling device may be in the form of a syringe and the return means may comprise a one-way valve for causing the amniotic fluid to flow through the cell filter in one direction only during the suction and pressure strokes of the piston of the syringe. Furthermore, the separating means may comprise a cell filter which may form part of the one-way valve. The one-way valve may be adapted to open during the suction stroke of the syringe so that amniotic fluid containing cell material may be sucked into the syringe cylinder through the hollow needle of the syringe. When the syringe piston is subsequently moved in the opposite direction through a pressure stroke the one-way valve closes so as to cause the cell-containing amniotic fluid to be forced through the cell filter arranged within the one-way valve, and the filtered amniotic fluid with a reduced cell content may then be returned to the amniotic cavity through the hollow needle. After the pressure stroke of the syringe piston cell material deposited on the cell filter and a small residual volume of fluid are left in the syringe. The residual amount of amniotic fluid may be used for alpha-fetoprotein determination. In order to avoid excessive sudden changes in pressure within the amniotic cavity small amounts of amniotic fluid may be sucked into the syringe at a time. Thus, the syringe piston may be moved through a number of short consecutive suction and pressure strokes. In this manner a substantial amount of cells may be removed from the amniotic fluid with a rather small physical influence of the pregnancy. When the filter material within the syringe has been filled with cells the resistance against the piston movements will increase and the sampling procedure may then be terminated. In case of amniocentesis at a late stage of pregnancy it may possibly be preferred to use a single relatively long suction stroke and a subsequent corresponding pressure stroke. After sampling the cells filtered from the amniotic fluid may be rinsed off the cell filter with culturing medium directly into a culturing flask whereby centrifugation of the sample is unnecessary. Alternatively, the cells may be cultivated directly on the filter as mentioned above.
Summarizing, the following advantages may be obtained by the method and apparatus according to the invention:
a) The collection of the samples can be carried out at an earlier stage of pregnancy due to the fact that an increased number of cells results in better cell growth.
b) The time required to obtain results is reduced considerably.
c) The system implies more independence as regards a specific stage of pregnancy at the time of sample collection, since the collection of sample may be continued until the resistance from the filter is increased so as to indicate that a sufficient number of cells has been collected.
d) The risk of inducing abortions due to the sample collection may be decreased, since only a very small proportion of the amniotic fluid is permanently removed from the amniotic cavity, whereby the conditions of pressure remain substantially unchanged in the amniotic cavity.
e) The risk of amniotic banding/amniotic prolapse leading to congenital malformations or abortions may be decreased by carrying out amniocentesis at the above early stage since the cell membranes are not adherent until the 10th-12th week of pregnancy.
f) The risk of developing lung complications in the newborns may also be reduced, since several studies have indicated a connection between the amount of amniotic fluid at the time of sampling collection and the risk of developing lung problems (Jo-Anne Finegan, 1984).
g) An increased proportion of living cells in the sample as compared to the presently used technique where only approximately 10% of the cells in the 16th week of pregnancy are alive. It is assumed that it is possible to obtain this larger proportion of living cells since the recirculation of amniotic fluid results in a larger fluidal flow around the needle point whereby an increased number of vital cells will be detached from the fetus and the membranes.
h) A reduction of non-successful samplings due to lack of cell growth since each individual sample will contain a large number of cells. (D. E. Rooney et al., 1989).
i) The costs involved in amniocentesis as compared to chorionic villi biopsy are lower.
j) An increased number of cell clones from which chromosomal analysis can be carried out will result in a more reliable chromosomal diagnosis as compared to the present situation where an answer in some cases is to be given on the basis of only 5 clones.
k) An improved diagnosis of chromosomal mosaicism due to the presence of more cells in the starting material.